(y.uu^har(crial ;uul ,^I^;il ^1Mlctaholism ; nut linVi ronmenial Biotechnology,I•asncem I atma (Ed)Copyright <>

1999 Narosa Publishing house, New Delhi. India

24. Seaweed Biotechnology

Padma NambisanPlant Biotechnology Unit, Department of Biotechnology,

Cochin University of Science and Technology, Cochin-682 022, India

Marine macroalgae or seaweeds consist of a group of diverse plants taxonomicallydistinguished into Chlorophyta (green seaweeds), Phaeophyta (brown seaweeds)and Rhodophyta (red seaweeds). Generally found growing on rocks, pebbles orother plants in the intertidal or subtidal regions of the sea coast, seaweeds have

been harvested for various purposes. A number of tropical seaweeds are eatendirectly (sea vegetables), including green algae

(Ulva, Enteromorpha,Monostroma, Caulerpa), brown algae (Dictyota, cladosiphon), and red algae

of iodine and bromine for the chemical industry [2]. In coastal areas throughoutthe world seaweeds such as Sargassunt have been used as manure. In India,Turhinaria and Hypnea

are used as manure for coconut plantations especiallyin coastal Tamilnadu and Kerala [3]. The major economic value of seaweeds is

however associated with the polysaccharide that certain red and brown algalspecies contain. Algins, carrageenans and agars have all achieved commercial

significance because of their food and industrial applications and are the basisof an estimated billion dollar global effort [4, 5]. While carrageenans and agars,

from which agaroses are derived by purification, are obtained from differentgenera of red algae, algins are obtained from a number of brown algae and are

present in all. A summary of sources, compositions, properties and more importantapplications of these polysaccharides is presented in Table I (modified from[I].

At present, most seaweeds are being harvested from naturally existing seaweedbeds, resulting in over harvested populations and slow regeneration which does

not meet the demand [7]. Several countries such as China, Japan, Korea, Chile,

Vietnam and India have adopted aquaculture mehods including pond culture,on bottom culture, net culture and raft culture to augument uroduction [91

Seaweed Biotechnology 237

for the improvement and better utilisation of algal resources has been mooted[9]. The present review in an attempt to collate efforts made in this direction.

1. Seaweed Tissue Culture

In higher plant systems almost all of the existing technology in genetic engineeringhinges on the availability of consistently reproducible protocols for the in vitroculture of cells, tissues or organs. A typical tissue culture cycle in higher plants,which are characterised by a high degree of organisation and differentiationinto tissues and organs, consists of hormonal induction of dedifferentiation ofcells of an excised plant part (explant) to form a callus, and the redifferentiationof cells of a callus to form a new plant. Higher plant cells are consequentlyconsidered to be totipotent [10], i.e., possessed of the genetic potential to directthe development of an entire plant from each cell of the organism.

Over the past couple of decades an increasing number of studies have dealtwith seaweed tissue culture. Chen and Taylors (1978) [11] work with Chondruscrispus is one of the first such studies. Most subsequent research has been basedon phycocolloid producing algae such as Gelidium, Laminaria and Graeilariaor edible species such as Porphyra, Eucheuma and Undaria. Because of theiraquatic habitat and unique chemical composition, seaweeds presentbiotechnologists with problems which are quite different from those confrontingworkers on higher plants. Polne-Fuller and Gibor (1978) [12] identify four specificcategories among the unique problems of algal tissue and cell cultivation asreproduced below:

A. Obtaining clean tissue that is free of other organismsThe surface of seaweeds are heavily infested by various microbial and largerepiphytes. Some of these organisms are embedded in cell walls and between theliving cells. This present an unique problem in seaweeds since their meristematiccells are frequently located on the surface and will be damaged upon applicationof chemical cleaning.

B. Dissociating the tissue to viable cells and protoplastsAlgal cell walls are composed of macromolecules which are more complex thancellulose (the major molecule in higher plant cell walls), thus different enzymesare required from those used for dissociation of higher plants. Few such enzymesare available commercially.

C. Inducing divisions and regeneration of isolated cells and protoplaststo form complete plants

Algae do not generally respond to the recognized hormones which effect thedevelopment and growth of higher plants. Unfortunately, little in known aboutspecific factors controlling algal growth and differentiation.

Table I Seaweed Polysaccharides

yaccharide Importantraw material (genera)

Composition

(Mixtures of Gelidium, Gracilaria Agarose = alternating 1,4 linked.e and Gelidiella a-D galactose and 3, i-anhydro)ectin) Pterocladia a-L-galactose backbone

Gracilariopsis (agarobiose) substit ited withPorphyra, Hypnea varying percent, ges of

methoxyl ester sulfat, and ketalpyruvate groupsAgaropectin = alter eating D-galactose and L-galas rose units.D-galactose can be s.rbstitutedby D-galactose-4-su phate, by4,6-0-(l carboxy ethy idene)-D-galactose in certain I retinalchain positions or e en by D-galactose 2,6-disulpl ate, whilepart of L-galactose can bereplaced by 3, 6 at hydro-L-galactose

'Alginates Macrocystis 1,4-linked a-L-gulu onic acidLantinaria and $-D-mannurcnic acidSar-gassum subunits in G.G, PI.M. andTurbinaria M.G. domains

°enans

fa-

kappa and iotaAlternating 1.3-linked a-D-galactose and 1,4linl ed 3,6-

anhydro-13-D-galacto ebackbone (carrabiose)substitiuted with var, ingpercentages of ester ! ulfate

Euchenna (cottonii), 4-sulfated on the ;alactose

Kappaphycus subunits (- 25% estc - sulfate)

(ah'arezii), Gigartina

(radula)

Euchema (spinosum) 4-sulfated op the ;alactose'subunits and 2-sulfated on the3,6-anhydrozalactos -- subunits(- 32% ester sulfate

Chondrus (crispus) Alternating 2-sulfates 1,3-linked

Gigartina (radula) a-D-glactosc and 2,6-disulfatcd1,4-linked /3-D galactosebackbone (minimal 3.6-anhydro-$-D-galactose) (- 35%

ester sulfate)

Important properties

Agaroses =* Gel aqueous solutions at low

concentrations* Form ion dependent

thermoreversible gelsControllable electroendo-simosis (EEO)

* Minimal non-specific proteinreactivity

* Significant degree of hysteresisAgars =* Ge}' aqueous solutions at low

concentration* Form thermoreversible gels* Relatively inest* Significant degree of hysteresis* Retain moisture* Resist hydrolysis by terrestrial

microorganisms

* Ammonium and alkali metalsalts are soluble in water,whereas free alginic acid andalkaline earth and Group IIIsalts are insoluble and can formgels

* Bind water

* Thicken aquous systems* Suspend solids* Bind moisture* Stabilize emulsions* Control flow and texture

properties of food systems* High protein reactivity-

strong interactions with milk

proteins

* Form strong rigid gels withpotassium and calcium ions

* Exhibit synergy with locustbean and konjac gums.

* Form elastic aqueous gels with

calcium ions* Exhibit synergy with locust'

bean gum and starch* Suspend particulates

* Non gelling aqueous system

viscosifter

Selected Applications

Matrices for:* Electrophoresis* Immunoassays* Microbial and cell culture* Chromatography* Immobilized Systems* Baking icings* Jelly candies* Canned meats* Dental impression media* Laxatives* Microbial culture matrix* Raw material for agarose

* Frozen foods to maintainstructure on thawing

* Baking icings* Salad dressings* Tahletting agent* Dental impression media* Textile sizin_r

Matrices for ilnriu hilizcd

* Frozen dessert stabilizers* Chocolate milk stabilizers* Texturizers for low-fat foods* Low calorie jellies* Toothpaste binders* Air freshners* Personal care products

* Pet foods

I]

240 NAMBISAN

1.1 Callus induction and plant regeneration in seaweed tissue culture

Callus formation is defined as the induction of a disorganised growth of cells

(or dedifferentiation) in a differentiated tissue as a result of wounding [131. The

structural and cellular organization of seaweeds differ significantly from higher

plants. The plant body or thallus in macro algae has evolved three main types of

cellular organization viz filamentous, pseudo parenchymatous and

parenchymatous. Although these three levels of organisation can be found among

members of all three division, most tissure culture work has been carried out

with pseudoparenchmatous and parenchymatous thalli (for review, see [14]).

The ease with which callus can be induced from an explant, or cultured upon

excision from the explants, seems to vary among members of three divisions.

Calluses rarely developed from mature sections of intact tissue of Chlorophycean

members but developed from the tissue sections in frequencies of 2-10%

(Laminariales) or 3-27% (Fucales) in Phaeophycean and 0.1-3% in Rhodophyceanmembers [15]. The potential for callus development in seaweeds in sometimesinfluenced by thallus thickness [14]: in the red and brown algae, development

of callus from intact explants has been mostly described in cases where the

thallus is made up of several cells, while in green seaweeds such as Enteromorphaand Ulva, the thallus is only one to two cell layer. In red seaweeds, callus mayarise from cortical cells (e.g. Gracilaria verrucosa [16]), but more often arisesfrom the medullary cells. No mention in made in literature of the potential for

callus induction in explants from different areas of the thallus, and regeneration

of calli into new plants has rarely been reported in red seaweeds [14], with theexception of Laurencia sp. [17]. Unlike calli from higher plants, that induced inred algae mostly cease growth upon excision from the explant [141

In brown seaweeds too, the medullary cells are associated with callus growth[18]. However unlike calli induced in red seaweeds, in some brown seaweeds,calli have been reported to resume growth upon excision from the explant (e.g.Sargassurn muticum [19]). Also, differences have been observed within differentparts of the thallus to develop a callus (e.g. blade > stipe > rhizoid) [20], possiblyrelated to the nutritional status of the cells involved [14].

The available information makes it difficult to explain why some calli cangrow independently of the explant while others cannot. In higher plant systemsinduction and maintenance of a callus, as well as regeneration of plants from acallus, is mediated by hormonesor plant growth regulators [21]. Although therehave been reports suggesting the involvement of plant growth regulators [22,23] in seaweeds, their presence has not yet been unequivocally established. Whileseaweeds are not known to respond to most hormones used in higher plant tissueculture, increased growth in Dictyota dichotoma cultures [24], or increased callusformation on explants of Laurencia sp. [25], have been reported upon additionof a water soluble extract of the respective alga. There is also sufficient evidenceto confirm the hypothesis [26] that under non-axenic conditions surfacemicroorganisms are able to supply growth promoting substances to their "hosts".As early as 1953, Ericsson and Lewis [27] demonstrated the transfer of vitamins

r pa'

Seaweed litotcehnolo,Gvv 241

from bacteria to algae. Chandramohan in 1971 [281 showed that bacteria living

on the surface of Enteromorpha intestinalis could synthesize the growth regulator

indole acetic acid (IAA) from tryptophan. Aclear case of bacteria contributing

to the 'normal' development of seaweeds was documented by Provasoli and

Pintncr 1291 and Tatawaki et al. [301, where Ulva and Monostroma developed

abnormally in axenic cultures grown in artificial seawater medium. The plants

regained normal morphology upon the addition of the bacteria which were isolated

from the plant. It has been speculated that the symbiotic association between

bacteria and algal host may induce the release of oligosaccharins from the plant

cell walls [14]. Oligosaccharins are often released in higher plants as a result of

fungal or bacterial attack and appear to be involved in defense as well as growth

regulation. [31].Another notable feature of seaweed tissue culture has been that the conditions

under which callus has been obtained are quite variable and, in gerneral, theoccurrence of a callus cannot be attributed to any specific set of experimentalconditions [14]. This suggests that internal factors inherent to the explants aremore important than the culture conditions employed.

Aguirre-Lipperheide et at. [14] in their review, opine that the apparent success

in obtaining callus using tissue culture techniques in seaweeds should beinterpreted cautiously. Two points to be considered should be the percentage ofcallus formed and the size of the calli. When compared to callus cultures ofhigher plants, seaweed calli are generally slow growing and small in size (-1-3mm) and the reaction to wounding often ceases after some weeks. Also, theoccurrence of calli in some seaweeds is sporadic and the percentages often verylow (< 2%) Consequently the suitability of callus derived from explants as abasis for cell suspension cultures, (iuL ^.u^.. °"d2' metnholiteproduction) is debatable [14]. It has been demonstrated that under appropriateconditions, seaweed polysaccharides can be produced by callus culture, including

agar from Pterocladia capillacea [32], but whether this would ever be a cost

effective production method for polysaccharides is doubtful.

1.2 Protoplast culture and somatic hybridisation in seaweedsProtoplasts (plant cells devoid of their cell walls) have been isolated and culturedin all the three classes of seaweeds. In the green seaweeds, the development ofcallus has seldom been reported, but regeneration into a whole new plant hasbeen reported in many cases. In the brown and red seaweeds regeneration ofprotoplasts into callus and into new plants is recorded in several species including

commercially important seaweeds such as Porphyra [19, 32], Gracilaria spp.

[9, 33] and Chondrus crispus [34] (for review, see [14]). It thus appears that this

technique is becoming well established in seaweeds. It is possible that thisbehaviour might be connected with the fact that a protoplast represents the lowestpossible unit of eukaryotic cell organization, and thus it is easier to reprogramgrowth from this point than in the case of a mass of walled cells with no cleardefinition of structure. At present, protoplasts are more suitable for establishing

--142 N:1,MBIS1N

cell suspension culture than callus derived from explants, and may have somepotential in secondary metabolite production, for example, cultured protoplastsof Kappaphvvcus alvarezii have been reported [35] to secrete carrageenanfragments. It has also been suggested that if generation of viable protoplaststhat form cell walls and divide could be achieved reproducibly and on a largescale, the protoplasts could be used as seed stock for macroalgal culture [6].

The fusion of cells or protoplasts permits the recombination of cytoplasmsand genomes of widely differing origins. During the last decade, the fusion ofterrestrial plant protoplasts has been routinely achieved through either chemicalmeans or by electrical impulses. Heteroplasmic fusions have been reportedin the green seaweeds, such as, Ulva spp. [36], Enteromorpha spp. [37] andMonostroma spp. [38], and in the red alga Porphyra spp. [39-44]. Fusion hasbeen brought about either by the use of polyethylene glycol [39-41 ] or by theelectrofusion technique [42-44]. Fusion efficiencies were found to depend onthe concentration and nature of reagents used to adjust the osmotic pressure ofthe medium [43].

2. Gene Mapping and Sequencing in SeaweedsA prerequisite for the genetic manipulation of an organism is an adequateunderstanding of genome structure, sequence and gene expression. In seaweeds,the presence of anionic polysaccharides which have similar properties to nucleicacids (they are precipitated by ethanol at about the same pH as nucleic acids)have complicated the isolation of DNA and RNA. Modified extraction protocolsto obtain DNA sufficiently pure for molecular applications such as restriction

I.... ,u,.,.,.." _ ;,uthcn blot hybridizations and amp lification via the--._ --a ypolymerase chain reaction (PCR), include CsCI-gradient ultracentrifugation [45],treatment with CTAB [46], hydroxyapatite binding [47] or purification onsepharose columns [48]. Since the viscous polysaccharides are released by grindingin liquid nitrogen, extraction by softening cell walls using lithium chloride in aprocedure that does not require grinding of tissues has also been reported [49].

Reports of mapping and sequencing of seaweed genomes are sparse. InGracilaria tenuistipitata the chloroplast ribosomal-protein encoding genes havebeen located, cloned and characterised [50]. A 1365 bp region around this genewas also sequenced and the gene order found to be identical to that detected inthe chioroplast DNA of liverwort, tobacco and maize. The plastid gene for therp 122 protein in G. tenuistipitata has also been isolated and sequenced [51 ].

It has been proposed that nucleic acid analysis including restriction analysisand detection of DNA sequence homologies could help in furthering ourunderstanding of speciation, phylogenetic and evolutionary biogeography withinthe algae [52-54], and in examination of heterosis [55]. In phylogenetic analysis,targets for sequence homology determination among the seaweeds have beenthe 5S ribosomal RNA [56], nuclear small subunit rRNA genes [53], the plastidrbcL gene coding for the larger subunit of ribulose-l, 5-bisphosphate carboxylase/oxygenase (RUBISCO) [57], nuclear genes encoding cytosolic and chloroplast

S,11ieerd HHinlrC1i0lo i'V 243

glyceraldehyde-3-phosphate dehydrogenases (GADPH)1581. gene amplificationproducts [59], the J3-tubulin gene [60], subunit 3 of cytochrome C oxidise 1611,and small subunit rRNA (SSUrRNA) fronr the mitochondria. 1621 Restrictionanalysis of plastid DNA fragments has been used to determine relatedness inred algal species [45]. In one study [63], this technique was used to prove thattwo macroalgae previously thought to be distinct species were actually identical.Nuclear DNA reassociation kinetics has also been used to determine inter- andintraspecific variations in selected agarophytes and carrageenophytes [64].

3. Genetic Engineering of SeaweedsRecombinant DNA technology has been effectively used for the improvementof crop plants due to the availability of suitable plasmids and expression vectors,

such as the Ti plasmid of Agrobacterium tuinefaciens [65], or viral vectors such

as Cauliflower Mosaic Virus [66], as well as methods of direct DNA delivery toeffect transformation such as electroporation, use of PEG, etc. [67], or usingmicroprojectiles [68]. One of the difficulties inherent in the application ofrecombinant DNA technology to seaweeds has been the development ofappropriate selection markers, vectors and efficient transformation systems.

Of twenty one red algal genera studied [45], five were found to contain circulards DNA plasmids. Some of these have been isolated and studied, and one 3.5

kbp plasmid from Gracilaria lemanefonnis was sequenced to reveal two potential

open reading frames. In this species, plasmids are present in a high copy numberper cell and may provide useful vectors for algal transformation. The DNA

sequence and structural organization of the GC2 plasmid from the agarophyte

hac been determined [47]. This 3827 bp circular plasmid

has one major open reading frame that generates a transcript and couia encoue

a 411 amino acid polypeptide.It has also been suggested that recombinant viruses particularly the large ds

DNA viruses that are known to infect eukaryotic algae can be used as

transformation vectors for marine macroalgae [69].

4. Commercial Implication of Seaweed BiotechnologyThe majority of commercial polysaccharide products have existed for years withthe same specifications and in general, companies are reluctant to alter thoselong accepted extracts and blends, and the raw materials from which they arerecovered [6]. Also, since many seaweed polysaccharides are destined for humanconsumption, they are therefore strictly regulated. For instance, in several countriesincluding USA, only carrageenans from specific seaweeds meet food ingredientregulation, and this discourages the quest for new species as sources of rawmaterial. Consequently, the immediate impact of biotechnology in commercial

cultivation of seaweeds could possibly be the following:

(1) In the generation of sufficient amounts of selected strains of marine

macroalgae for cultivation : Currently about 25% of cultivated material is used

244 NAM131SAN

for seeding the next crop. If consistently reproducible methods of generation ofplants from protoplasts are developed, these could be used as seedstock [6].

(2) In the use of DNA sequences or molecular probes to prove equivalence ofraw material: Because the species acceptable as sources have been identifiedby name in the regulation , alternative, perhaps more abundant or easily cultivated,species are often excluded, even though polysaccharide extracts from them maybe essentially identical [6]. Acombination of molecular biology, refined analyticalmethods such as NMR spectroscopy and traditional morphological taxonomywill possibly produce a more appropriate list of acceptable raw materials [70].

(3) In the development of engineered polysaccharides in the non-consumablesmarkets: Genetically altered algae will present the same regulatory problemsthat surround genetically engineered crop plants whose products are targettedfor human consumption. However since various polysaccharides are requiredfor industrial purposes, a cost-effective alternative may be obtained fromgenetically engineered seaweeds.

AcknowledgementsThe author is grateful to the Indian Council of Agricultural Research for a researchproject entitled "Improvement of marine red and brown algae for enhancedproduction of agar-agar and algin using tissue culture techniques" which providedthe necessary funding for work in this field. The encouragement and guidancereceived from Dr. N. Kaliaperumal, Scientist, CMFRI, Mandapam Camp, TamilNadu, is gratefully acknowledged.

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